Laboratory Techniques To Study Neurodegeneration

Question 1

What is TDP-43?

Answer 1

RNA binding protein
TAR DNA binding protein 43 kilo Daltons

Question 2

Which neurons die in ALS?

Answer 2

Upper motor neurons
Lower motor neurons

Question 3

What percentage of ALS patients have familial gene mutations?

Answer 3

10%

Question 4

What condition overlaps with ALS?

Answer 4

FTD

Question 5

What gene is the biggest genetic cause of ALS and FTD?

Answer 5

C9orf72

Question 6

What is linkage analysis?

Answer 6

Used in families with identified disease

Analyses which portions of chromosomes have been inherited throughout the family and comparing that with the pattern of inheritance of disease

Question 7

What are the two potential mechanisms of TDP-43 pathophysiology?

Answer 7

Aberrant behaviour in the cytoplasm (gain of function)
Loss of activity in the nucleus (loss of function)

Question 8

Which animals do not require ethics approval for laboratory work?

Answer 8

Fruit flies (drosophila melanogaster)

Question 9

How does CRISPR/Cas9 work?

Answer 9

Guide RNA is used to match the target area
The RNA molecule is complexed to Cas9
Complex is introduced to the cell
Cas9 protein inserts the new sequence at the target area

Question 10

What does the western blot assay do?

Answer 10

Antibody is used to tag and quantify amount of protein

Question 11

What kind of misregulation leads to increased TDP-43 concentration?

Answer 11

Negative feedback disruption
TDP-43 regulated its own expression by destroying TDP-43 RNA
Disruption in this leads to increased RNA and TDP-43 Protein

Question 12

How are fibroblasts and blood cells turned into pluripotent stem cells (iPSCs)?

Answer 12

Induction using Yamanaka factors
Oct3/4, Sox2,Klf4, c-Myc

Question 13

What is direct differentiation?

Answer 13

Going directly from one cell type to another without becoming an iPSC
e.g. fibroblast to neuron
Retains epigenetic markers

Question 14

How do antisense oligonucleotides work?

Answer 14

Prevent expression of RNA into protein

Binds to the target mRNA, recruits RNAase which catabolises the mRNA

Question 15

Which diseases have current ASO trials ongoing?

Answer 15

Tau in Alzheimer’s
SOD1 in MND
C9orf72 in MND
Huntingtin in HD

Question 16

Which ASO treatment is licensed in the UK?

Answer 16

Nusinersin for SMA

Question 17

What is the utility of histopathology in studying neurodegeneration?

Answer 17

Identification of protein aggregates

Question 18

If a protein, peptide residue, or DNA sequence is highly conserved through species, what might that signify?

Answer 18

The protein is vital to basic functioning
The further back the conservation, the more important the protein is likely to be

Question 19

What is the lifetime risk of ALS?

Answer 19

1 in 300
(Same as MS)

Question 20

What is the role of TDP-43 in the nucleus?

Answer 20

RNA splicing